Open AccessMutations of the Yku80 C Terminus and Xrs2 FHA Domain Specifically Block Yeast Nonhomologous End Joining
Author(s) -
Phillip L. Palmbos,
James M. Daley,
Thomas E. Wilson
Publication year - 2005
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.25.24.10782-10790.2005
Subject(s) - ku80 , ku70 , non homologous end joining , biology , dna ligase , rad50 , dna repair , saccharomyces cerevisiae , ddb1 , dna , genetics , homologous recombination , ubiquitin ligase , microbiology and biotechnology , dna binding protein , yeast , gene , ubiquitin , transcription factor
The nonhomologous end-joining (NHEJ) pathway of DNA double-strand break repair requires three protein complexes inSaccharomyces cerevisiae : MRX (Mre11-Rad50-Xrs2), Ku (Ku70-Ku80), and DNA ligase IV (Dnl4-Lif1-Nej1). Much is known about the interactions that mediate the formation of each complex, but little is known about how they act together during repair. A comprehensive yeast two-hybrid screen of the NHEJ factors ofS. cerevisiae revealed all known interactions within the MRX, Ku, and DNA ligase IV complexes, as well as three additional, weaker interactions between Yku80-Dnl4, Xrs2-Lif1, and Mre11-Yku80. Individual and combined deletions of the Yku80 C terminus and the Xrs2 forkhead-associated (FHA) domain were designed based on the latter two-hybrid results. These deletions synergistically blocked NHEJ but not the telomere and recombination functions of Ku and MRX, confirming that these protein regions are functionally important specifically for NHEJ. Further mutational analysis of Yku80 identified a putative C-terminal amphipathic α-helix that is both required for its NHEJ function and strikingly similar to a DNA-dependent protein kinase interaction motif in human Ku80. These results identify a novel role in yeast NHEJ for the poorly characterized Ku80 C-terminal and Xrs2 FHA domains, and they suggest that redundant binding of DNA ligase IV facilitates completion of this DNA repair event.